JPS60500684A - Method for determining antigen active amino acid chain - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for determining antigen active amino acid chain The present invention relates to a method for detecting or determining antigenic activity of amino acid chains in proteins. related.

As used throughout this specification, "antigenic activity" refers to amino acids that specifically bind to antibodies. refers to a chain of amino acids that elicits or stimulates the production of antibodies. (this latter chain is also called the "immunogen").

As is already well known, antigens are usually unfamiliar to humans and animals. proteins, which can elicit the formation of antibodies by humans or animals. Antibodies respond to the presence of this macromolecule in humans or animals. It is a protein synthesized by

Antibodies have a specific affinity for the macromolecule that elicited their synthesis, that is, the antigen; The specificity of antibodies is usually determined by antigenic determinants. one or more special positions on a macromolecule called nt) It is based on chains.

The first object of the present invention is to prepare selected proteins such as for example Antigen determination of antigens that can yield antibodies that protect against certain clinical diseases such as The objective is to detect or determine the amino acid chains that constitute the fixed group.

According to the invention, within a known amino acid chain of a protein or part of a protein, A method of detecting or determining an antigenically active amino acid chain is provided.

This method consists of the following steps.

1. Synthesize multiple peptides. Each of these peptides has a known amino acid chain This peptide consists of a chain of multiple amino acids, corresponding to a chain within, and this peptide has overlapping It has a chain of amino acids.

2. Each of these peptides can be used as an antibody against a protein or a part of a protein. bring into contact with.

3. Between each of these peptides and the antibody, the peptide has antigenic activity. Detecting or determining whether or not there is an antigen that indicates the presence or absence of an antigen.

As mentioned above, peptides synthesized according to this method contain overlapping amino acid chains. peptide, i.e. at least one amino acid is present at one end of the chain of the other peptide. and at least one amino acid is added to the other end of the chain. , the remaining amino acids are common to both chains and one peptide interacts with the other. linked.

The amino acids in such overlapping chains are such that each sequence is a protein or protein as described above. or to correspond to a chain within a known amino acid chain of a part of a protein. selected.

The method of the invention allows a given antibody to identify a chain of amino acids that has specific antigenic activity. The concept of identifying exactly, and as a result of this, among all possible combinations Identification using the high specificity of antibodies to identify amino acid chains of specific antigenic determinants It is based on the concept that the antigenic determinants of

Confirmation of the chain of amino acids representing the active antigen or antigenic determinant of the immunogenic protein Many publications have been published with this goal in mind.

However, even with recently available methods, active molecules within protein molecules cannot be Confirming amino acid chains is a long and boggling process.

According to the present invention, a screen using peptides having overlapping chemical chains as described above This is a method to detect chains of amino acids that are active as antigens.

Antigenic determinants are generally believed to consist of a chain length of approximately six amino acids. , the chain of amino acids produced according to the first step of the invention is therefore preferably It is a chain of six amino acids.

However, according to the method of the present invention, a chain of five amino acids in length can be This results in significantly fewer reactions that can be easily detected in screening procedures. Although it seems unlikely, it is important to note that the invention is not limited to chains of six amino acids. You should understand.

Chains of nine or more amino acids in length are probably unnecessarily long and therefore Such a chain is not desirable at present. However, these chains also apply to the present invention. It is included within the broad scope of.

In practicing the present invention, proteins believed to have the antigenic determinant of interest are The amino acid chain of the protein or protein portion is predetermined.

The amino acid sequences of many proteins are already known and can also be (recombinant) Recent methods such as DNA technology are It is useful as a method to rapidly elucidate the chains of proteins whose chains are not yet known. Ru.

The importance of the present invention lies in the fact that antigenic determinants of proteins can be easily synthesized from short amino acids. This is because it can be confirmed as an acid chain. Furthermore, all contiguous antigenic determinants of interest You can check it.

This information has the necessary selectivity to be used in the diagnosis of clinical diseases in humans and animals. This will be of immeasurable benefit in the design of drugs that

Unfortunately, knowledge of the antigenic determinants associated with a particular pathogen is also important, as is the case with conventional vaccines. To produce peptide vaccines that have no harmful side effects and are useful in protecting against diseases. Required.

The invention can also be used to design specialized therapeutics for the body's receptors. Ru.

Use of the above method in determining antigenic determinants within a protein or portion of a protein. An example of this is as follows.

1. First, the protein or proteins obtained, for example from publication data. For the known amino acid chain of the part, the first six amino acids (in this example , the order ψ) assuming that all synthesized peptides are six amino acids long starting from the amine terminus or starting from the carboxy terminus and converting this into “sequence 1”. '.

Then start from the second amino acid and include the seventh amino acid. Let it be the central amino acid chain "sequence 2".

Drop one amino acid from this -terminus and add one amino acid to the other end of the known chain. By continuing to add and drop the tamper All hexapeptides contained within a substance or part of a protein A amino acid chain is obtained.

The total number of such chains is the number of amino acids forming the protein or part of the protein. 5 less than the actual number.

2 Each chain determined as above was then synthesized.

Suitable synthetic methods include the well-known methods for peptide synthesis, commonly referred to as: Includes methods that are used.

(al solution phase method; or (bl solid phase method; for example, Merrifield technology: Lugrin, 1. (Marglin, A.) and Merrifield, R. , (Merrifield, R, B,), 7 = Near Le Review Op Biochemistry (Ann, Red.

Bicochem, ) 39, 841-866 (1970).

However, preferably the synthesis of amino acid chains is carried out using polyethylene as a solid phase support. or using a polymeric material such as polypropylene, on which at least one A vinyl monomer containing two functional groups is graft-polymerized to form polymer chains on a carrier. It is carried out using a solid-phase method.

The functional groups of these polymeric chains then react to form primary or secondary amine groups. , which amino groups are then sequentially reacted with amino acid residues to an appropriate degree to obtain the desired A synthetic peptide is formed.

The carrier is preferably a solid polymer rod having a diameter of about 4m11L and a length of about 5Qmm. It has the shape of

Many such rods have dimensions that are suitable for enzyme-linked immunosolvent assays (euzyme-1 inch). ked immunosorbentassay (E LI S A)) It is held in a suitable holder with a 12×8 grid corresponding to the dimensions of the standard plate used.

3. Depending on the selection of operations for the synthesis of each peptide, the peptide is If the solid support is already attached to a suitable support or the preparation of the assay step is carried out on a suitable solid support. connected in time.

4. If the carrier supports the synthesized peptide, use a microtiter plate (m1crotit plate). (replate) or similar device space (well) K, and then each Known chains are commonly used to detect the presence or absence of antigen-antibody reactions within each space (weld). screened against known antibodies using the method of

Examples of these methods include enzyme-linked immunosolvent assays (ELISAs) and radioactive radio immunoassay.

RIA).

Suitable antibodies may be purchased as commercially available antisera or may be well-known. Therefore, it is produced in a suitable host animal.

Conveniently, for the purpose of organizing the synthesis of the six lengths of amino acid chains mentioned above, A computer-based management program is used.

Record the final peptide essence for each position in each microtiter plate. The sequence of syntheses that can occur simultaneously is determined based on compatible reaction conditions. Help you decide.

Finally, such a computer management program is the most advanced at the antibody screening stage. It can also be used with a folded program for final evaluation.

As set forth in more detail in the Examples, the method outlined in the above steps: Amino acid chain active as an antigen in the FMDV protein VPI Used to confirm strands.

Of course, the present invention applies to hepatitis as well as other proteins with known linkages. Hepatitis B virus surface antigen Other viruses, such as en).

It is similarly applicable to the determination of antigenic determinants of proteins.

The well-known indirect ELISA technique uses a It is preferably used for the final detection or determination of the presence or absence of an antigen-antibody reaction.

The use of this assay is limited to final tests such as anti-human IgG, anti-human IgG, etc. It is necessary to produce an antiserum against the pile body species used for this purpose.

Such preparation is then carried out using horseradish peroxidizer. In combination with enzymes such as enzymes, it provides the necessary test drug for final testing.

Indirect FLISA testing is performed using techniques well known in the field. be exposed.

When the method of the invention is carried out by a screening test of the type described above, The results obtained for a particular antibody belong to one of the following categories.

fa) There is no positive reaction for any of the produced amino acid chains, i.e. If completely negative.

(b) When only a single amino acid chain reacts.

This result is an ideal case.

(C) Case where many amino acid chains react.

One of the following two reasons is expected for this observed result.

One reason is the reaction with antisera containing mixed antibody groups, where each antibody is associated with a different antigen. Reacts at the site.

Another reason is the reaction of immunogenic sites with overlapping amino acid chains.

In the case of the first reason, a more useful test to determine the antigenic active amino acid chain is performed. Need more.

Of course, once the antigen-active amino acid chain reacts with a given antibody, When detected or determined, this information can be used in various diagnostics and in the production of vaccines. It can be used for.

In fact, when the antigenically active amino acid chain corresponding to a particular clinical disease drug is determined. This suggests that it may confer protection against the disease and elicit the desired antibody response. one or more synthetically derived amino acids, one or more suitable amino acids; It can lead to the production of vaccines consisting of acid chains.

More detailed features of the invention are illustrated by way of example only in the examples below.

Example 1 Nucleites translated by Klunn, Neu, () (urtz, C,’) et al. Nucleic Ac1d Re5earch 9 , 1919-1931 (1981), VPI (FMDV, type O ), the 213-amino acid chain of The hexapeptide units were placed on a polyethylene carrier in the same orientation and as shown in Figure 1. It was synthesized using two lengths of amino acids as spacers.

A polyethylene rod immersed in 6 volumes of acrylic acid aqueous solution was irradiated with IMrad. γ-rays were irradiated. [Mul Ier -5c hulte, l), ), Hol, <ter,! 7,1 I, (Horste r, F, A, ') + Polymer-hyurte: / (polymer 1 3ulletin) 7, 77-8] (1982)].

Using conventional methods of solid-phase peptide chemistry [Erichnon, B., W. (E. r1ckson, B, W,) + Merrifield.

Merrifield, R,B, “The Proteins ('pl]e proteins) - Volume 2, pages 255-257, Akatemi Nok Press (Academic Press), = -L-Yoke (Ne wYork) <1973)], No t-butyl oxy7carboni Lu L-IJ resin methyl ester is added to polyethylene via the N-amine group on the side chain. It was bonded to polyacrylic acid (PPA, ).

Then, [BOC-alanine is attached] to complete the peptidic space. I set it.

aS) substitution on the carrier, labeling NH2-lysine (OMe)-PPA with CI4 Determined by reacting with butyric acid and found to be 8 to IOn mol/rod. I found it.

Continue following standard solid phase peptide synthesis, as directed by the chain to be synthesized. Amino acids were added.

The final coupling reaction is completed and the t-butyloxycarbonyl (BOC) protecting group is removed. After that, the terminal amino group was treated with didinotylformamide (DMF)/to-11 ether. Acetylated with acetic anhydride in a min mixture.

All synchrohexol-luposiimide-mediated coupling reactions are performed with N -Carried out in DMF in the presence of hydroxybenzotriazole.

The following side chain protecting groups were used.

About s / oni / l serine, anubartic acid, glutamic acid and tyro-7 is O-benzyl; is tosyl; /Stin is 4-methylbenzyl 2 and and histidi/1-benzyloxi7carponylamide 2.2.2- In trifluoroacetic acid.

Removal of side chain protecting groups was achieved by dilution of boron[·lis() in trifluoroacetic acid for 90 minutes at room temperature. Trifluoroacetate) [Press, G.I., ( Pless.

J,), Bauer, W, (Bauer, W,), Angewante Chemie 85, 142 (1973 )reference〕.

included in the synthesis as a control after hydrolysis in HCI/propionic acid. From the analysis of the chain, it was confirmed that campling occurred at each step.

Prior to testing by ELISA, the rods and camped peptides were separated from the phosphate pan. Washed several times with saline solution (PBS).

B. Hexapeptide test Antigen profile (pro file) is a numerical value giving the position within the VPI chain of the N-terminal amino acid heftide. Above, the obtained ELIS is shown in FIG. 2 as a vertical line proportional to A extinction.

The antisera used to obtain the different profiles shown in Figure 2 are as follows: It is as follows.

(al and (bl) two different anti-intact beers particles, type O Anti-scald virus particles used in (b) after absorption with C1 purified complete virus; Type 01゜ (di antiviral static subunit (5ubunit), type 01; (e ) Anti-VPI, type 01 and +f+ Anti-scald virus particles, type C0゜rod and camplated peptide (R CP) to test the activity of each of the defined antisera described above. An enzyme-coupled immunosolvent assay was used.

RCPS for 1 hour at 37°C to block nonspecific absorption of antibodies. 0% horse serum, 10 thiovalbumin and 1 cytowin (’l’w) in PBS. een) -80 and was destroyed in advance.

Antiblood diluted to 1//io with preincubation mixture After overnight incubation at 4°C in supernatant, 0.05% Tween Washed three times with -80/PBS.

Horseradish Peruvian diluted to /so,ooo in the pre-incubation mixture for 1 hour at 37°C. After reaction with a suitable JgG immunoglobulin camplated with oxidase, Repeat again with KPBS/) Tween to remove excess bonding. and washed.

The presence of the antibody was determined using a developer solution (orthophenylenediamine 40ml9.hydrogen peroxide 20ml). 1 dissolved in 100 mJ of phosphate buffer, pH 5.0) for 45 minutes. The developed color was detected at 420 nm by Titerchik multiski I read it on Camera 7 (Titertek Multiscan).

After testing, the peptides were dissolved in 0.1% 2-mercaptoethanol and 0.1% sulfur. Washed three times with 8M urea solution containing sodium dodecyl acid and removed all bound The cells were washed several times with PBS to remove traces of antibody.

RCPs can further be used to test different antisera.

Anti-scald virus particle serum contains 50% of rabbit inactivated and purified viruses! i by immunization in 70 g of complete 70 g.

The rabbit bled for 3-4 weeks after a single injection.

Antivirus, subunit (5ubunit) serum (rabbit) destroyed with acid 10 μg of purified virus was administered three times at intervals of 3 to 4 weeks, initially using Freund's Immunizations were performed with complete adjuvant, then incomplete Freund's adjuvant. Manufactured by

Polypeptide vP1 is a protein obtained from urea-disrupted and purified viruses. From the protein mixture, the isoelectric center method (5o-electric focusing) separated by [Barteling, Nis, G.I. S, J, ), Wagenaar, F, (Wagenaar, F, >, Gi Lukens, A., L.

Gielkens, A, L, J, LJ, Gen, V irol, 62357-361 (1982)].

After elution from the gel with 8M urea and dialysis against PBS, the antiserum was purified against 128. was generated as described above.

Antiserum against scan (C) was prepared from purified virus (1500 μg of complete virus). were incubated with 1 ml of serum at 4°C for 72 hours), so after absorption, the scan (This was used for bl, and all the antibodies bound to the virus were removed by centrifugation.) Removed.

Confirmation of viral particles bound to C2 antigen peptide The four anti-scald viruses tested above scan (al and +b+ are the activity patterns found) It reached its peak in .

Large quantitative differences in responses to the same antigen production have been previously reported. . However, these scans capture possible variability in antibody composition between sera. I'm emphasizing it.

From the scan (al, (bl and (C1) tests, peptide numbers 146 and Antibodies active against and 147 are present in whole anti-intact virus serum, but not with purified virus. Not present after absorption.

These same antibodies were not observed in the anti-burn serum of scan fd) and Only a small amount was present in the anti-VPI serum of (e).

The finding of some activity in the anti-VPI serum indicates that the isolated protein , are thought to show immunological activity, albeit weakly [Kreit, T., C., (Kl. eid, D-J, et al., 5science, 214, 112 5-1129 (1981)].

However, other anti-VPI sera have also been tested and have strong activity at position number 148. However, it should be noted that there was no activity at positions 146 and 147. It is possible.

6 The overlap between scan+b+ and scanFC+ (absorption relative to non-absorption) is determined by the peptide number. In addition to the activity of peptide numbers 146 and 147, peptide numbers 5, 6 and 206 This indicates that a decrease in activity has also occurred.

Among these, the activity of numbers 5 and 6 was the highest in all antiscald virus sera tested. The activity of number 206, on the other hand, was always present.

From these results, among the sequences found to be active, numbers 146 and Both in 147 are hexapeptides and, consistent with other observations, number 2 It is concluded that the distribution is much smaller than the position in 06, and it constitutes the main position. .

Gly-ASJ)-Leu-Gln-Val-Leu (G-D-L-Q-V -L) and ASI) -Leu-Gln-Val-I, eu-Aha (D -L-Q-V-L-A) However, regarding the position of number 146.147, we can distinguish between the following two possibilities. I can't do it.

One possibility: the active element is five amino acids long, i.e. two ASI)- I, eu-Qln-Val-Leu　(D-L-Q　-V-L)K common sequence.

Another possibility: the active element is seven amino acids long, i.e. two hexapepeptides. Gly-Asp-I, eu-Gln-Val-I, eu-Ala (G-D-L-Q-V-L-A) combination.

A. Production of hexapeptide Office anticulnational de eviscétiques (Office '1n International des Epizootics) Baclaff.

Given by Bachrach, H, L, et al. VPI (FMDV Taifu AI. or A6.) no 2127:'/acid The chain was subdivided into 207 hexapeptides.

These hexapeptides are modified by replacing the arginine side chain with p-methoxybenzenesulfonyl It was synthesized in the same manner as in the above example except that it was protected with a group.

B. Hexapeptide test The antigenic profile of the hexapeptide is shown in FIG.

The antiserum used to produce this contour is as follows.

Shin) Anti-scald virus particles, type A1゜(bl) Purified complete FMDV type After absorption with AIO (anti-scald virus particles used in al. Hexapeptide testing and serum preparation are essentially the same as described in Example 1. .

Confirmation of viral particles bound to C6 active element As used in Example 1 For the same reason, the following hexapeptides are the antigenic determinants of FMDVO 8-type. It can be concluded that it occupies a major position.

Gly -Asp-Leu -Gly-8er-11e (G-D-L-G-8 -I) and ASI) -Leu-Gly-8er-11e -Ala Example 1 As in the case of FMD virus, type 01, described in therefore, the active chain is five amino acids long, i.e. As p-Leu-Gly-8er-11e (D-L-G-8-I) or seven Amino acid length, i.e. Gly-Asp-Leu-Gly-8er-1 1e -Ala (G-D-L-G-8-I-A). discussed.

Example 3 Journal of Virology ) 46.311-316 (1983) Roberton, Ench.

vpl, given by Robertson, H, L, et al. (FMDV type C,) 210 amino/acid chain is subdivided into 205 hexapeptides. Ta.

These hexapeptides were synthesized as described in Example 2 above.

B. Hexapeptide test The antigenic profile of the hexapeptide is shown in FIG.

The antiserum used to create the profile is as follows.

fat anti-scald virus particle type C3 (b) purified complete FMDV type C After absorbing in 1 (al ward ward ward international search report C1ted in 5earch Patent Family Member sepport

Claims (1)

[Claims] 1 Antigen-active amino acids within known amino acid chains of proteins or protein moieties A method for detecting or determining acid chains, the method comprising the following steps. 1. Multiple amino acids, each peptide corresponding to a chain within a known amino acid chain multiple peptides, each of which has an overlapping amino acid chain. synthesize, 2. Contacting each of the peptides with an antibody against the protein or protein moiety let me, 3 Each of the peptides and the antibody indicating whether the peptide has antigenic activity detect or determine the presence or absence of an antigen-antibody reaction between 2 Claim 1, in which the plurality of peptides overlap at one amino group The described method for determining antigenic active amino acid chains. 3. A patent claim in which each of the plurality of peptides is a chain of five to nine amino acids The method for determining the antigenic active amino acid chain according to item 2. 4. A claim in which each of the plurality of peptides is a chain of six amino acids 4. The method for determining an antigenic active amino acid chain according to item 3. 5. Each of the plurality of peptides is synthesized on a solid phase carrier, and bound to the solid phase carrier. Each Vepti held The antigen according to claim 1, on which the subsequent steps of the method are carried out. How to determine active amino acid chains. 6. Claim No. 6, wherein the solid phase support is in the form of a pin or rod made of a solid polymer. 5. The method for determining an antigenic active amino acid chain according to item 5. 7. The solid phase carrier is selected from the group consisting of polyethylene and polypropylene. It is a polymer material that has been grafted onto this polymer material to form a polymer on a carrier. Patents containing vinyl monomers containing at least one functional group giving rise to side chains A method for determining an antigenic active amino acid chain according to claim 5 or 6.